The Caliciviridae are a family of positive-strand RNA viruses and consist of four genera designated: (1) Norovirus (with species Norwalk virus); (2) Sapovirus (with species Sapporo virus); (3) Vesivirus (with species, feline calicivirus and vesicular exanthema of swine virus); and (4) Lagovirus (with species rabbit hemorrhagic disease virus and European brown hare syndrome virus). Norwalk virus is the prototype strain for the genus Norovirus and was discovered by LID researchers in 1972. The noroviruses are the major cause of nonbacterial epidemic gastroenteritis that occurs in family, school, institutional, or community-wide outbreaks, affecting all age groups. The noroviruses are also an important cause of sporadic, acute gastroenteritis in infants and young children. The human noroviruses are genetically diverse and cannot be grown in cell culture, which has been a continuing research obstacle. [unreadable] [unreadable] A major goal of this laboratory is the development of control strategies for the caliciviruses (predominantly the noroviruses) associated with gastroenteritis. In order to accomplish this goal, basic knowledge of the epidemiology, immunology, and replication strategies of these viruses is needed. In collaboration with Dr. Purcell, we continued studies of Norwalk virus infection and immunity in chimpanzees. This animal model offers a promising alternative to human volunteer studies in the initial development and testing of norovirus vaccine candidates. A proof of principle study that evaluates the efficacy of Norwalk virus-like particles (VLPs) as a vaccine is in progress. As a second approach in the development of an animal model for vaccine development, we initiated collaboration with Dr. Linda Saif (OSU). Dr. Saif obtained stools from two individuals with acute gastroenteritis and administered one of the stool filtrates containing a GII.4 norovirus (strain HS-66) to gnotobiotic calves by the oral route (Souza et al., J. Virol. 2008; 82:1777). The infectious dose was reported as 5.4 x 106 genome equivalents. All five calves showed evidence of mild diarrhea. We have received viral RNA purified from the original inocula, as well as stool specimens from inoculated calves in order to compare our quantitative methods. We generated a complete consensus sequence of the HS-191 genome (which was closely-related, but higher-titered than HS-66) and constructed a recombinant baculovirus that expressed HS-191 VLPs. Dr. Saif will evaluate these VLPs as a vaccine candidate in calves.[unreadable] [unreadable] The vesiviruses have a broad host range, and previous studies in Dr. Linda Saif's laboratory found evidence for its presence in mink. Because we wanted to address the question of zoonotic transmission of vesiviruses, we decided to further characterize this vesivirus and compare its antigenic and genetic relatedness with other caliciviruses. Three mink calicivirus (MCV) strains (designated MCV 9, 13 and 20) were provided by Dr. Saif. The viruses were adapted to efficient growth in cell culture by serial passage in Vero cells three times, and then subjected to three rounds of plaque-purification. Virions were purified by isopynic centrifugation and specific hyperimmune sera were raised in guinea pigs. Western blot analysis showed evidence for the presence of shared antigens between MCV and feline calicivirus (FCV), but no cross-reactivity was observed with human or murine noroviruses. Sequence analysis of the MCV genome showed that these viruses were most closely related to canine calicivirus (strain 48). However, the MCV sequences formed a distinct branch within the genus, consistent with its designation as a separate species in the genus Vesivirus. We expressed the mature VP1 of the MCV9 strain and it self-assembled into VLPs, enabling the development of a diagnostic ELISA for the detection of antibodies in human and animal sera. The availability of this vesivirus in the laboratory will facilitate comparative studies among the cultivatable caliciviruses.[unreadable] [unreadable] We continued work focused on the basic mechanisms of calicivirus replication. We developed an in vitro nucleotidylylation reaction to study the mechanism by which the norovirus RNA-dependent RNA polymerase modifies the viral VPg. Studies focused on replication may lead to the development of novel approaches to block virus infection.